Marker proteins for diagnosing liver disease and method of diagnosing liver disease using the same

ABSTRACT

Using the protein chip technology, biological samples such as sera are subjected to proteome analysis. Thus, a protein which is a human fibrinogen α-E chain decomposition product and has a molecular weight of 5,900, a protein which is an apolipoprotein AII decomposition product and has a molecular weight of 7,800, and a protein which is an apolipoprotein AI decomposition product and has a molecular weight of 28,000, each showing an increase or a decrease with the habit of drinking, are newly found out. By detecting or quantifying these proteins, a liver disease in a subject such as one having a problem of drinking can be diagnosed at the early stage.

This application claims priority under 35 U.S.C. §119 of Japaneseapplication No. 2002-371959, filed on Dec. 24, 2002.

TECHNICAL FIELD

The present invention relates to a plurality of serum proteins whichhave been found to increase or decrease with the habit of drinking andto be hence utilizable as marker proteins for diagnosing liver disease,as a result of proteome analysis of serum samples using the protein chiptechnology; and a method for diagnosing the probability of the onset ofa liver disease, the liver disease, the prognosis of the liver disease,or the like in, for example, a problem drinker, by detecting orquantifying the above-mentioned proteins.

BACKGROUND ART

The first step to the diagnosis of an organ trouble caused by alcohol isto know an accurate drinking history, but alcohol dependence is called adenying disease, namely, an inveterate drinker does not accuratelyreport his (or her) alcohol intake in all ages and countries. Therefore,an objective marker for substantiating the alcohol intake is necessary.A marker for the habit of drinking most commonly measured is γ-GTP(GGT). However, even if a drinker has a high GGT level, the GGT leveldoes not always correlate with the degree of seriousness of livertrouble or the cumulative alcohol intake of the drinker. In addition,the change of GGT level after alcohol drinking is dependent onindividuals and there are a considerable number of so-callednonresponders who show no GGT increase even after drinking a largeamount of alcohol.

On the other hand, by a cause other than drinking, GGT is oftenincreased also in a person having no drinking habit, such as a personhaving fatty liver due to fatness or a person who habitually takes acertain medicine. Guidance is often given, for example, in a hospitalfor complete physical examination so that a person having a high GGTlevel may be hastily judged to be a drinker. Therefore, chain-deficienttransferrin (CDT) has been developed by investigators in north Europefor examination complementary to the examination with GGT, and itsusefulness is emphasized in European and American references. But, inthe case of the result obtained for Japanese, CDT as a marker ofdrinking permits detection of only about 10% of the GGT nonresponders.

Acetaldehyde, the first metabolite of ethanol is so reactive that itforms various acetaldehyde adducts of various proteins. For example,attempts have been made to detect an acetaldehyde-hemoglobin adduct byHPLC or the like. There is also an adduct that is expected to be aninteresting marker capable of permitting estimation of alcohol intake inthe past, such as HbAlc in the case of diabetes. But, such an adduct hasnot been put to practical use because of its low sensitivity.

The habit of drinking is one of two major causes of chronic livertroubles. As to liver cirrhosis cases in Japan, the percentage of casesdue to only alcohol itself is considered to be only 10 to 15%. This,however, is data obtained mainly in university hospitals and the like.Considering the presence of alcoholics in a number estimated at morethan 2,000,000, it is speculated that there are many latent patientswith alcoholic liver trouble who have no chance to get a medicalexamination in a medical institution. In addition, since the habit ofdrinking is a factor of the exacerbation of cerebral hemorrhage,hypertension, gout and the like, early and accurate screening of problemdrinkers is very important. However, as described above, there is nomarker having decisive sensitivity and specificity among existingso-called markers of drinking, and hence searching for a novel marker isdesired.

A general method for comprehensive expression protein analysis istwo-dimensional protein electrophoresis, but this method isdisadvantageous in the detection of low-molecular weight proteins orpeptides. In recent years, a protein chip technology comprising acombination of surface enhanced laser desorption ionization (SELDI) anda time-of-flight mass spectrometer has been developed by CiphergenBiosystems Inc., USA and has been begun to be clinically used for, forexample, detecting a novel tumor marker. Therefore, comprehensive searchfor a novel marker by the utilization of such a proteomics technique andthe like is desired.

DISCLOSURE OF THE INVENTION

The present invention is intended to find a novel marker capable ofpermitting early and accurate screening of the liver diseases of problemdrinkers or the like, establish a system for measuring the marker andtake advantage of the marker in medical treatment.

The present inventors have done intensive research on the above problemand consequently have accomplished the present invention. That is, thepresent inventors have succeeded in identifying novel serum proteinseach of which shows an increase or a decrease with the habit ofdrinking, by the use of serum samples periodically collected fromalcoholics hospitalized for giving up drinking. The present inventorshave found that these serum proteins are utilizable as marker proteinsfor diagnosing liver disease, whereby the present invention has beenaccomplished.

Accordingly, the present invention relates to a marker protein fordiagnosing liver disease selected from a protein which is a humanfibrinogen α-E chain decomposition product and has a molecular weight of5,900 (5.9 kDa protein), a protein which is an apolipoprotein AIIdecomposition product and has a molecular weight of 7,800 (7.8 kDaprotein), a protein which is apolipoprotein AI and has a molecularweight of 28,000 and variants of these proteins which have the samefunction as that of the proteins as a marker protein for diagnosingliver disease.

In addition, the present invention relates to a method for diagnosingthe probability of the onset of a liver disease, the liver disease orthe prognosis of the liver disease by detecting or quantifying any ofthe above-mentioned marker proteins for diagnosing liver disease in asample obtained from a patient who is suspected to have the liverdisease.

Further, the present invention relates to a novel protein having theamino acid sequence shown as SEQ ID NO: 1 in the sequence listing, orits variant having the same function as that of said protein as a markerprotein for diagnosing liver disease, which variant is a protein having90% or more homology with said amino acid sequence or a protein havingan amino acid sequence formed by deletion, substitution or addition ofone or more amino acid residues in the amino acid sequence shown as SEQID NO: 1.

Still further, the present invention relates to a novel protein havingthe amino acid sequence shown as SEQ ID NO: 2 in the sequence listing,or its variant having the same function as that of said protein as amarker protein for diagnosing liver disease, which variant is a proteinhaving 90% or more homology with said amino acid sequence or a proteinhaving an amino acid sequence formed by deletion, substitution oraddition of one or more amino acid residues in the amino acid sequenceshown as SEQ ID NO: 2.

Still further, the present invention relates to a method for measuringany of the above-mentioned proteins or their variants by immunoassay bythe use of an antibody against the protein or variant to be measured.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the measurement results obtained from the serum of apatient with alcoholic liver trouble by the use of SAXII chips byutilizing Protein Chip System available from Ciphergen Biosystems Inc.From the decrease in height of the peak, it can be seen that the levelof the 28 kDa protein (apolipoprotein AI) decreases in the serumgradually with the lapse of time in the order of the level at the timeof admission, the level after hospitalization for 1 week and the levelafter hospitalization for 3 months.

FIG. 2 shows the measurement results obtained from the serum of apatient with alcoholic liver trouble by the use of WCXII chips in thesame manner as in FIG. 1. It can be seen that the blood levels of (1)the 5.9 kDa protein and (2) the 7.8 kDa protein increase with treatmentwith the lapse of time from the blood levels at the time of admission.

FIG. 3 shows the measurement results obtained from the serum of ahealthy person by the use of WCXII chips in the same manner as inFIG. 1. The blood levels of (1) the 5.9 kDa protein and (2) the 7.8 kDaprotein are definite high levels, and comparison between FIG. 2 and FIG.3 indicates that these proteins are decreased by the disease.

FIG. 4 shows the result of electrophoresis of the 7.8 kDa protein andthe 28 kDa protein by SDS-PAGE. It can be seen that the samples containthe proteins of interest, respectively.

FIG. 5 shows the mass spectrometry value and HPLC data of synthetic 5.9kDa protein. It can be seen that the mass spectrometry value agrees withthe theoretical value.

FIG. 6 shows the result of comparing data for the synthetic 5.9 kDaprotein with data for the serum of a patient who does not drink, by theuse of WCXII protein chip arrays by utilizing Protein Chip Systemavailable from Ciphergen Biosystems Inc. Both of them show a peak at 5.9kDa and they show the same behavior.

FIG. 7 shows the result of measuring the height of a peak at 5.9 kDa inthe case of a serum sample from each of healthy persons and patients,who are various in alcohol intake, by the use of WCXII protein chiparrays by utilizing Protein Chip System available from CiphergenBiosystems Inc. It was found that the height of the peak decreasesdepending on the alcohol intake.

FIG. 8 shows the result of measuring absorbance by the use of an EIAmethod (a sandwich ELISA method) by using samples containing variousconcentrations of the 5.9 kDa protein. The axis of abscissa refers tothe concentration of the 5.9 kDa protein and the axis of ordinate toabsorbance measured. The absorbance increases depending on theconcentration of the 5.9 kDa protein. That is, this result indicatesthat the concentration of the 5.9 kDa protein in the samples may bemeasured by the EIA method.

MODE FOR CARRYING OUT THE INVENTION

The present invention is explained below in further detail.

The proteins that have been found to be utilizable as marker proteinsfor diagnosing liver disease by the present invention are a proteinwhich is a human fibrinogen α-E chain decomposition product and has amolecular weight of 5,900 (hereinafter referred to as the 5.9 kDaprotein), a protein which is an apolipoprotein AII decomposition productand has a molecular weight of 7,800 (hereinafter referred to as the 7.8kDa protein), and a protein which is apolipoprotein AI and has amolecular weight of 28,000 (hereinafter referred to as the 28 kDaprotein). The 5.9 kDa protein, the 7.8 kDa protein and the 28 kDaprotein are proteins having the amino acid sequences shown as SEQ IDNOs: 1, 2 and 3, respectively, in the sequence listing.

The individual proteins are explained below. The 5.9 kDa protein is ahuman fibrinogen α-E chain decomposition product, comprises 54 aminoacids and has a theoretical molecular weight of 5904.2. The 7.8 kDaprotein is a human apolipoprotein AII decomposition product, comprises68 amino acids and has a theoretical molecular weight of 7753.8. The 28kDa protein is apolipoprotein AI, comprises 243 amino acids and has atheoretical molecular weight of 28078.8. As to the 5.9 kDa protein andthe 7.8 kDa protein among the above proteins, the present invention haselucidated their presence in blood and their clinical significance inliver troubles and the like for the first time. The 5.9 kDa protein andthe 7.8 kDa protein are novel proteins and novel marker substances.Apolipoprotein AI, the 28 kDa protein is a known protein, its clinicalsignificance in lipid metabolism has been established, and its clinicalmeasurement has heretofore been carried out.

The 5.9 kDa protein, 7.8 kDa protein and 28 kDa protein, which have beenfound to function as marker proteins for diagnosing liver disease by thepresent invention, are not limited to proteins having the amino acidsequences shown in the sequence listing but may be variants of theseproteins which similarly function as marker proteins for diagnosingliver disease. That is, it should be sufficiently considered that the5.9 kDa protein, 7.8 kDa protein and 28 kDa protein are very liable tobe degraded by various endo- and exoproteases particularly in blood andtissues, resulting in changes of the total length of amino acids and thelength of the sequence. In producing a recombinant protein, an aminoacid variation capable of changing the antigenicity as slightly aspossible should, of course, be given in order not to decrease theefficiency of expression. Therefore, there may also be used variants ofthe 5.9 kDa protein, 7.8 kDa protein and 28 kDa protein of the presentinvention which are proteins having 90% or more homology with the aminoacid sequence of each of the proteins of the present invention (here,the term “homology” means the sameness of amino acids). The amino acidsequence of any of the variants may have a length changed by 15% orless, and the present invention also includes such a variant when itfunctions as a marker protein for diagnosing liver disease. The variantsare preferably proteins having 95% or more homology, more preferably 98%or more homology. The homology of the amino acid sequence may be lookedup in well-known software, for example, software obtained by adopting asa principle the reference method described in the method of Wilber, W.J., Lipman, D. J., et al. (Proc. Natl. Sci. USA, 80, 726-730, 1983). Inaddition, GENETYX (Software Development Co., Ltd.) or the like iscommercial general-purpose software and is easily utilizable.

The variants of the 5.9 kDa protein, 7.8 kDa protein and 28 kDa protein,which are the marker proteins for diagnosing liver disease of thepresent invention, may also be variants which are proteins having anamino acid sequence formed by deletion, substitution or addition of oneor more amino acid residues in each of the amino acid sequences shown asSEQ ID NOs: 1, 2 and 3 and have the same function as that of the abovethree proteins as marker proteins for diagnosing liver disease. As suchvariants, there are exemplified proteins obtained by the modification ofless than 10%, preferably less than 5%, more preferably less than 2%, ofthe amino acid residues in the original amino acid sequence. Themodification of the amino acid residues may be introduced as amino acidvariation by a genetic technique generally known to those skilled in theart. The present invention also includes variants obtained by well-knownmodification such as posttranslational modification, phosphorylation,acetylation, sugar chain addition, or the like.

Diagnosis of liver diseases becomes possible on the basis of the markerproteins for diagnosing liver disease found by the present invention andexplained above. That is, the probability of the onset of a liverdisease, the liver disease or the prognosis of the liver disease may bediagnosed by detecting or quantifying the above-mentioned markerproteins for diagnosing liver disease in a sample obtained from apatient who is suspected to have the liver disease.

As the sample usable in the present invention, there are exemplifiedserum, plasma, blood and urine collected from the patient who issuspected to have the liver disease.

All methods known at present may be adopted for detecting or quantifyingthe marker proteins for diagnosing liver disease of the presentinvention. The methods include, for example, mass spectrometry method,immunoassay method, electrophoresis method, liquid chromatography (LC)method and gas chromatography (GC) method.

As the mass spectrometry method, a method using a laserdesorption/ionization-time of flight-mass spectrometer (LDI-TOF MS) isexemplified. As the laser desorption/ionization-time of flight-massspectrometer, there may be exemplified surface enhanced laserdesorption/ionization-time of flight-mass spectrometers (SELDI-TOF MSmethod) and matrix-assisted laser desorption/ionization-time offlight-mass spectrometers (MALDI-TOF MS method).

For example, when SELDI-TOF MS method is adopted, Protein•Biology•SystemII•Mass•Spectrometer (Ciphergen Biosystems, Inc.) developed by CiphergenBiosystems, Inc. may be used. This machine is based on a protein chiptechnology comprising a combination of SELDI (surface enhanced laserdesorption ionization) and a time-of-flight mass spectrometer. Thedetails of the machine are disclosed in International Publication No. WO01/25791 A2, JP-A-2001-28122 and the like. Usually, in SELDI-TOF MSmethod, a sample is pretreated, adsorbed on a chip and then loaded on aSELDI-TOF MS mass spectrometer. When the sample is serum, it ispreferable to remove albumin from the system by using an adsorbent foralbumin or washing the system with a buffer solution until thepossession of electric charge by albumin owing to the ion exchange chipis ceased.

The protein chip used in such a method is not particularly limited solong as it can adsorb the marker proteins for diagnosing liver diseaseof the present invention. As the protein chip, there may be exemplifiedchips (referred to also as chemical chips) in which functional groupshaving hydrophobicity or affinity for proteins (e.g. ion exchangeproperties) have been modified, and chips (biochemical chips) having anantibody against the protein of interest immobilized thereon.

As another mass spectrometry method, a mass spectrometry method usingESI method (electrospray ionization) is exemplified. In the case of ESImethod, it is often preferable to load a sample subjected topretreatment such as protease treatment on a mass spectrometer connecteddirectly to a separating means such as high performance liquidchromatography.

As the immunoassay method, there may be exemplified an immunoassaymethod in which a heretofore known protein is measured by preparing apolyclonal or monoclonal antibody against any of the marker proteins fordiagnosing liver disease of the present invention. Such an immunoassaymethod includes enzyme immunoassay method (EIA method),immunoturbidimetry method (TIA method), latex immuno-agglutinationmethod (LATEX method), electrochemiluminescence method, fluorescencemethod and the like. An immuno-chromatography method and a method usingtest paper are also effective. All of these methods are generally knownto those skilled in the art and these generally known methods may beadopted as they are.

As the antibody usable in the above-mentioned immunoassay method, thereare exemplified polyclonal or monoclonal antibodies prepared bygenerally used methods. These antibodies may be obtained by using apurified protein derived from human blood, specifically, the 28 kDaprotein, the 7.8 kDa protein or the 5.9 kDa protein as an immunogen (anantigen). Although these proteins for preparing the antibodies may beobtained from human blood by purification, they may be obtained also bychemical synthesis by adopting a well-known peptide synthesis technique.Besides these antigens, proteins produced by cultured cells may also beused as an antigen. In addition, the employment of a full-lengthrecombinant protein prepared by genetic engineering, its variant, or aportion of the recombinant protein or the variant is also a well-wornmeasure, and this measure may be utilized.

The monoclonal antibodies are produced by hybridomas obtained byimmunizing an animal with an immunogen such as any of theabove-exemplified various antigens, for example, the 28 kDa protein, the7.8 kDa protein and the 5.9 kDa protein, namely, the marker proteins,and then fusing antibody-producing cells derived from the spleen or thelike with myeloma cells.

The hybridomas may be obtained by the following method. That is, theantigen (e.g. the marker protein) obtained as described above is mixedwith a well-known adjuvant such as Freund's complete or incompleteadjuvant, aluminum hydroxide adjuvant, pertussis adjuvant or the like toprepare an adjuvant liquid for sensitization, and this liquid isadministered to an animal (e.g. a mouse or a rat) subcutaneously in theabdominal cavity or intravenously in the tail, in several portions atintervals of 1 to 3 weeks to immunize the animal. Although the amount ofthe antigen for the sensitization is usually chosen in the range of 1 μgto 100 mg, it is preferably about 50 μg in general. Although the numberof immunizing operations is generally 2 to 7, various methods are known.Subsequently, antibody-producing cells derived from the spleen or thelike are fused with myeloma cells or the like in a test tube. As to amethod for the fusion, the fusion may be carried out by the use of apoly(ethylene glycol) (PEG) by the method of KÖhller and Milstein(Nature, 256, 495, 1975) which is already per se well known. The fusionmay be carried out also by the use of Sendai virus or by anelectrofusion method.

As to a method for selecting hybridoma capable of producing an antibodycapable of recognizing the marker protein, from the fused cells, theselection may be carried out as follows. That is, the hybridoma isselected from colonies formed by cells surviving in HAT medium and HTmedium in limiting dilution of the fused cells. When an antibody againstthe marker protein is contained in the supernatant of the culture mediumfor any of the colonies formed by the fused cells seeded into a 96-wellplate or the like, a clone capable of producing a monoclonal antibodyagainst the marker protein may be selected by an ELISA method in whichthe supernatant is placed on an assay plate having the marker proteinimmobilized thereon, and after the reaction, a secondary labeledantibody such as anti-mouse immunoglobulin-HRP labeled antibody isreacted with the above-mentioned antibody. As the labeling substance ofthe labeled antibody, there may be used enzymes (e.g. alkalinephosphatase), fluorescent substances, radioactive substances and thelike besides HRP. Screening of specific antibodies against the markerproteins, respectively, may be conducted by carrying out, as a control,ELISA using an assay plate having only BSA bonded thereto as a blockingagent, simultaneously with the above-mentioned ELISA. That is, a cloneis selected which is positive in any of plates having the markerproteins, respectively, immobilized thereon, and is negative in theELISA method using BSA.

For example, hybridomas CN-1 and CN-2 established by the presentinventor are clones capable of recognizing human 5.9 kDa proteinspecifically and are preferable examples. Hybridomas CN-1 and CN-2 weredeposited as follows in Patented Organism Deposition Center (IPOD),Industrial Technology General Research Institute (IndependentAdministrative Corporation), Chuo-dairoku, Higashi 1-1-1, Tsukuba City,Ibaraki Prefecture, Japan 305-8566: hybridoma CN-1 was deposited as areceipt number IPOD FERM BP-08564 on Dec. 12, 2003 and hybridoma CN-2was deposited as a receipt number IPOD FERM BP-08565 on Dec. 12, 2003.

The hybridoma is cultured on a medium usually used for cell culture,such as α-MEM, RPMI1640, ASF, S-clone or the like, and the monoclonalantibody may be recovered from the supernatant of the medium. Thefollowing is also possible: after a nude mouse, an animal from which thehybridoma is derived, is previously treated with pristane, cells areintraperitoneally injected into the animal to cause accumulation ofascites, and the monoclonal antibody is recovered from the ascites. As amethod for recovering the monoclonal antibody from the supernatant orthe ascites, a conventional method may be adopted. There are exemplifiedsalting-out with ammonium sulfate, sodium sulfate or the like,chromatography, ion exchange chromatography, and affinity chromatographyusing protein A.

The 28 kDa protein, 7.8 kDa protein or 5.9 kDa protein in a sample maybe accurately measured by the use of the monoclonal antibody accordingto the present invention purified by the above method. As a method formeasuring the 28 kDa protein, 7.8 kDa protein or 5.9 kDa protein in asample by an EIA method, there may be adopted a per se well known methodusing one or more monoclonal antibodies against the marker protein. Anexample of the method is described below. At first, the monoclonalantibody (antibodies) against the marker protein is directly orindirectly bonded to a per se well-known solid phase (e.g. apolystyrene, polypropylene, polycarbonate, polyethylene, nylon orpolymethacrylate) by utilizing physical bonding, chemical bonding oraffinity. The amount of the sensitizing antibody ranges usually from 1ng to 100 mg/ml. A sample is added to the monoclonal antibody(antibodies) bonded to the solid phase by physical bonding, chemicalbonding or affinity to carry out the reaction. After a definite periodof the reaction, the solid phase is washed and a corresponding secondarylabeled antibody (e.g. an anti-28 kDa protein secondary labeledantibody, an anti-7.8 kDa protein secondary labeled antibody or ananti-5.9 kDa protein secondary labeled antibody) is added to carry outthe secondary reaction. The solid phase is washed again and DABcolor-producing substrate or the like is added thereto to carry out thereaction. When HRP is used as the labeling substance, a known substratesuch as DAB, TMB or the like may be used. The labeling substance is notlimited to HRP. As the labeling substance, there are exemplified allrecognizable substances including not only enzymes but also labelingmetals (e.g. gold colloid and europium), various chemical or biologicalfluorescent substances (e.g. FITC, Rhodamine, Texas Red, Alexa and GFP)and radioactive substances (e.g. ³²P and ⁵¹Cr).

The marker proteins may be measured also by a method other than theabove-mentioned immunoassay method, such as an electrophoresis method,liquid chromatography (LC) method, gas chromatography (GC) method or thelike. These methods are also generally known to those skilled in the artand these generally known methods may be adopted as they are.

By the method explained above, the probability of the onset of a liverdisease, the liver disease or the prognosis of the liver disease may bediagnosed by detecting or quantifying the marker proteins for diagnosingliver disease in a sample obtained from a patient who is suspected tohave the liver disease. When the diagnosis method of the presentinvention is practiced by the above-mentioned mass spectrometry, thediagnosis may be carried out also by analyzing the pattern of a spectrumobtained with a mass spectrometer. The diagnosis method of the presentinvention permits diagnosis of the probability of the onset of a liverdisease in a habitual drinker or a problem drinker, diagnosis of a liverdisease caused by drinking, such as hepatitis, liver cirrhosis or thelike, and diagnosis of a usual liver disease. Furthermore, it alsopermits diagnosis of, for example, the progress of treatment of liverdisease. The diagnosis method of the present invention is particularlysuitable for diagnosis of alcoholic liver troubles, alcohol dependenceand the like.

The present invention is illustrated in further detail with reference tothe following examples, which should not be construed as limiting thescope of the invention.

EXAMPLE 1

Identification of a marker protein for diagnosing liver disease by theUse of SAXII protein chip arrays

Using the sera of patients from whom informed consent had been obtained,a novel marker for liver trouble in the sera was searched for by the useof SAXII protein chip arrays (Ciphergen Biosystems, Inc.). The SAXIIchip refers to a strong anion exchange chip and is characterized in thatit binds thereto a negatively charged substance in a sample. The sera ofthe patients with alcoholic liver trouble immediately after admission,those 1 week after abstinence and those 3 months after abstinence andthe sera of normal persons were used as samples.

(1) Method

A method for experimental operation of the protein chip array is brieflydescribed below. Each serum sample was diluted 10-fold with a 8M urea(SIGMA)/1% CHAPS (SIGMA) solution. After on-ice treatment for 10minutes, the serum sample was further diluted 10-fold with 50 mM Tris(SIGMA) buffer (pH 9.0) and centrifuged at 4,000 rpm for 5 minutes, andthe supernatant was used as a diluted sample. An experiment was carriedout by attaching the SAXII chip to a bioprocessor. The bioprocessor is aperforated plastic adaptor for simply forming three-dimensional wells ona metal chip. By such a method, a large volume of the diluted sample maybe applied.

At first, 150 μL of 50 mM Tris buffer (pH 9.0) was added to the chiphaving wells formed thereon, and the chip was washed on a shaker for 5minutes. After this procedure was carried out twice, 100 μL of thediluted sample previously obtained was added to the chip and shaken atroom temperature for 20 minutes to be reacted with the chip. Then, thediluted sample was removed, and 150 μL of 50 mM Tris buffer (pH 9.0) wasadded to the chip, followed by washing on a shaker for 5 minutes. Thisprocedure was repeated three times. Thereafter, the chip was washedtwice with 400 μL of distilled water and then removed from thebioprocessor. After the chip dried, each spot having a protein adheredthereto was surrounded with PAPen (Zymed) and 0.5 μL of a saturatedsolution of sinapinic acid (Ciphergen Biosystems, Inc.) in 50%acetonitrile (Wako) and 0.5% TFA (Wako) was added twice to the spot. Theprotein chip arrays thus prepared were read with Protein•Biology•SystemII•Mass•Spectrometer (Ciphergen Biosystems, Inc.).

(2) Results

FIG. 1 shows typical measurement data obtained from the serum of apatient with alcoholic liver trouble at the time of admission. In thisdata format, the axis of abscissa may refer to the molecular weight of aprotein in each sample detached from the SAXII protein chip, and theaxis or ordinate may refer to a peak reflecting the amount of an analytewhich has arrived at the detector at the aforesaid molecular weight.Therefore, it was found that as is clear from FIG. 1, a peak due to aprotein having a molecular weight of 28 kDa was observed in the data forthe patient with alcoholic liver trouble at the time of admission butwas hardly observed after the admission. Accordingly, it was found thata liver disease may be diagnosed by using this 28 kDa protein as anindication.

EXAMPLE 2

Identification of marker proteins for diagnosing liver disease by theuse of WCXII protein chip arrays

Next, using exactly the same samples as in Example 1, novel markers forliver trouble in the sera were searched for by the use of WCXII proteinchip arrays (Ciphergen Biosystems, Inc.). The WCXII chip refers to aweak cation exchange chip and is characterized in that it binds theretoa positively charged substance in a sample.

(1) Method

A method for experimental operation of the protein chip array is brieflydescribed below and is substantially the same as in Example 1. Eachserum sample was diluted 10-fold with a 8M urea (SIGMA)/1% CHAPS (SIGMA)solution. After on-ice treatment for 10 minutes, the serum sample wasfurther diluted 10-fold with 50 mM sodium acetate (SIGMA) buffer (pH6.5) and centrifuged at 4,000 rpm for 5 minutes, and the supernatant wasused as a diluted sample. An experiment was carried out by attaching theWCXII chip to a bioprocessor. At first, 150 μL of 50 mM sodium phosphatebuffer (pH 6.5) was added to the chip having wells formed thereon, andthe chip was washed on a shaker for 5 minutes. After this procedure wascarried out twice, 100 μL of the diluted sample previously obtained wasadded to the chip and shaken at room temperature for 20 minutes to bereacted with the chip. Then, the diluted sample was removed, and 150 μLof 50 mM sodium phosphate buffer (pH 6.5) was added to the chip,followed by washing on a shaker for 5 minutes. This procedure wasrepeated three times. Thereafter, the chip was washed twice with 400 μLof distilled water and then removed from the bioprocessor. After thechip dried, each spot having a protein adhered thereto was surroundedwith PAPen (Zymed) and 0.5 μL of a saturated solution of sinapinic acid(Ciphergen Biosystems, Inc.) in 50% acetonitrile (Wako) and 0.5% TFA(Wako) was added twice to the spot. The protein chip arrays thusprepared were read with Protein•Biology•System II•Mass•Spectrometer(Ciphergen Biosystems, Inc.).

(2) Results

FIG. 2 shows typical measurement data obtained from the serum of apatient with alcoholic liver trouble at the time of admission. FIG. 3shows measurement data for normal persons. The following was found: asis clear from FIG. 3, the peaks at a molecular weight of 5,900 Da (the5.9 kDa protein) and a molecular weight of 7,800 Da (the 7.8 kDaprotein) are high in the data for the normal persons, but these peaksare hardly observed in the data shown in FIG. 2, i.e., the data for thepatient with alcoholic liver trouble immediately after admission. Theheights of the peaks due to the 5.9 kDa protein and the 7.8 kDa proteinincrease with treatment and clearly indicate the effect of thetreatment. It was found that these proteins may be used as markerproteins for diagnosing liver disease.

EXAMPLE 3

Identification of the 28 kDa Protein

(1) The 28 kDa protein found by the SAXII protein chip experiment waspurified from a serum sample with FPLC Pharmacia LKB (Amersham PharmaciaBiotech AB) by the use of a HiTrap Q column under the followingconditions: 50 mM Tris buffer (pH 9.0) and a flow rate of 2 ml/min.Thus, the 28 kDa protein of interest could be purified as asubstantially single fraction. This fraction was confirmed as follows byelectrophoresis. The fraction was mixed with 2× sample buffer (0.25 MTris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.01% BPB and 10%β-mercaptoethanol) in the ratio of 1:1 and treated at 90° C. for 2minutes. The fraction thus treated was used in the electrophoresis. Theelectrophoresis was carried out at 10 mA by the use of 15 to 25%polyacrylamide gradient gel (Perfect NT Gel System Products).

(2) As shown in each of the lanes 4 and 5 in FIG. 4, a band wasconfirmed at a position corresponding to 28 kDa, by Coomassie BrilliantBlue staining using Coomasie Tablet R-350 (Phast GI Blue R).

(3) Then, the band of the gel was excised and peptides were separated byan In-Gel digestion method. In brief, the piece of the gel obtained byexcision was washed twice and then treated overnight with trypsin at 35°C. Thereafter, the sample treated with trypsin was purified by reversedphase HPLC. As to the purification conditions, gradient elution with0.1% TFA and 0.09% TFA 90% acetonitrile was carried out by the use of aTSK gel ODS-80Ts QA (TOSOH) column.

The internal amino acid sequences of the resulting 28 kDa proteinfragments were determined. The amino acid sequence of the 28 kDa proteinis shown as SEQ ID NO: 3 in the sequence listing. As the result of aminoacid sequencing of the 28 kDa protein fragments, the 28 kDa protein wasfound to be human apolipoprotein AI on the basis of the internal aminoacid sequences of the 28 kDa protein fragments.

(4) Then, apolipoprotein AI levels were measured for the same serumsamples as used in the chip experiments by an immunoassay method usingthe known autoanalyzer described in Example 6, to obtain the resultsshown in Table 1. From the immunoassay results, it was found thatapolipoprotein AI levels were clearly reduced after treatment in thesamples obtained from the patients who had gained effect from thetreatment. On the other hand, the height of the peak due to the 28 kDaprotein (apolipoprotein AI) observed as a result of the protein chipexperiment was also reduced by the treatment, namely, it reflected theeffect of the treatment. Thus, it was found that there is a very highcorrelation between the result obtained by the immunoassay method andthe result of the protein chip experiment, indicating that the height ofthe peak is utilizable for analyzing morbidity.

EXAMPLE 4

Identification of the 7.8 kDa Protein

(1) Like the 28 kDa protein, the 7.8 kDa protein found by the WCXIIprotein chip experiment was also purified from a serum sample with FPLCPharmacia LKB (Amersham Pharmacia Biotech AB) by the use of a HiTrap CMSepharose FF column under the following conditions: 50 mM ammoniumacetate buffer and a flow rate of 2 ml/min. Thus, the 7.8 kDa protein ofinterest could be purified. A fraction in which the 7.8 kDa protein hadbeen confirmed by the protein chip experiment was confirmed byelectrophoresis as follows. The fraction was mixed with 2× sample buffercontaining SDS, in the ratio of 1:1 and treated at 90° C. for 2 minutes.The fraction thus treated was used in the electrophoresis. Theelectrophoresis was carried out at 10 mA by the use of 15 to 25%polyacrylamide gradient gel.

(2) As shown in each of the lanes 2 and 3 in FIG. 4, a band wasconfirmed at a position corresponding to 7.8 kDa, by Coomassie BrilliantBlue staining. As in Example 3 for the above-mentioned 28 kDa protein, afraction containing substantially only this protein of 7.8 kDa wasconcentrated and then subjected to SDS-PAGE, and the 7.8 kDa band ofinterest was excised from the gel and peptides were separated by anIn-Gel digestion method. This method is the same as in Example 3. Inbrief, the piece of the gel obtained by excision was washed twice andthen treated overnight with trypsin at 35° C. Thereafter, the sampletreated with trypsin was purified by reversed phase HPLC. As to thepurification conditions, gradient elution with 0.1% TFA and 0.09% TFA90% acetonitrile was carried out by the use of a TSK gel ODS-80Ts QA(TOSOH) column.

(3) The resulting 7.8 kDa protein fragments were subjected to amino acidsequencing and found to be derived from human apolipoprotein AI, on thebasis of the internal amino acid sequences of the 7.8 kDa proteinfragments. However, the theoretical molecular weight of full-lengthhuman apolipoprotein AI is 11432.4. That is, the 7.8 kDa protein wasfound to be a human apolipoprotein AI decomposition product. The aminoacid sequence of the 7.8 kDa protein is shown as SEQ ID NO: 2.

EXAMPLE 5

Identification of the 5.9 kDa Protein

(1) The 5.9 kDa protein found by the WCXII protein chip experiment waspurified from a serum sample with FPLC Pharmacia LKB (Amersham PharmaciaBiotech AB) by the use of a HiTrap CM Sepharose FF column under thefollowing conditions: 50 mM ammonium acetate buffer and a flow rate of 2ml/min. Thus, the 5.9 kDa protein of interest could be purified. Afraction having a high content of the 5.9 kDa protein was checked againby a protein chip method, concentrated and then further purified by HPLC(TOSOH). The purification conditions were as follows: a Sephasil proteinC4 column (Amersham Pharmacia Biotech AB), an acetonitrile gradient, and1 mL/min.

(2) The fraction was checked again by a protein chip method,concentrated by freeze-drying and then subjected to amino acidsequencing. As a result of the amino acid sequencing of the purified 5.9kDa protein, this protein was found to be a human fibrinogen α-E chaindecomposition product. That is, since the molecular weight offull-length human fibrinogen α-E chain is 72488.3, the 5.9 kDa proteinwas found to be a human fibrinogen α-E chain decomposition product. Theamino acid sequence of the 5.9 kDa protein is shown as SEQ ID NO: 1.

In addition, in order to confirm the accuracy of the amino acid sequenceof the 5.9 kDa protein, a protein having this amino acid sequence waswholly and chemically synthesized. The molecular weight of thechemically synthesized protein having 54 amino acids was 5904.1 whichagreed with the theoretical value (FIG. 5). Furthermore, this syntheticprotein was compared with an actual sample. An experiment was carriedout by the use of exactly the same WCXII protein chip arrays (CiphergenBiosystems, Inc.) as in Example 2. In the experiment, the syntheticprotein was reacted in a concentration of 100 ng/mL as a sample andcompared with the actual sample. The results are shown in FIG. 6. As aresult, a peak due to the synthetic protein showed a behavior coincidingwith a peak due to the 5.9 kDa protein in the serum sample. By theseresults, it was confirmed that the 5.9 kDa protein in the serum had theamino acid sequence shown as SEQ ID NO: 1.

EXAMPLE 6

Comparison Between Diagnosis in Patients with Alcoholic Liver TroubleBased on the Marker Proteins for Diagnosing Liver Disease of the PresentInvention and that Based on Conventional Markers for Hepatitis

(1) Method

Measured values for conventional markers for hepatitis were obtained byusing the same sera as used above, i.e., the sera of the 16 inpatientswith alcoholic liver trouble immediately after admission, those after 1week of hospitalization and those after 3 months of hospitalization.Measurement with an autoanalyzer was carried out as follows. AST, GGT,TG, Apo AI and Apo AII were measured with Hitachi 7150 analyzer(HITACHI). As reagents, N-assay L GOT (AST) (Nittobo), N-assay L γ-GTP-H(GGT) (Nittobo), N-assay TG L (TG) (Nittobo), N-assay TIA Apo AII-H (ApoAI) (Nittobo) and N-assay TIA Apo AII (Apo AII) (Nittobo) wereindividually used. FDP and FDP-E were measured with LPIA-S500(Diayatron) by using predetermined parameters. In this measurement, LPIAFDP latex and LPIA FDP-E latex (Teikoku Hormone MFG. Co., Ltd.) wereused as reagents. For comparison, the results of measurement usingprotein chips are expressed by numeral values on the basis of peaks. Theunits of the numeral values are arbitrary units.

(2) Results

Table 1 shows the following items in the common sera of patientshospitalized with alcoholic liver trouble: the levels of biochemicalmeasurement markers for clinical examination (AST, GGT and TG), thelevels of immunological measurement markers (Apo AI, Apo AII, FDP andFDP-E) and the results of measurement of the marker proteins for liverdisease of the present invention by a protein chip method. In the firstand second columns (the “FDP-E 5,900 Da” column and the “Apo AII 7,800Da” column) from the right of Table 1, there are shown measured valuesobtained by measuring the 5.9 kDa protein and the 7.8 kDa protein, i.e.,the marker proteins for diagnosing liver disease of the presentinvention, by a method using the same protein chips as in Example 2. Inthe sixth column (the “Apo AI” column) from the right of Table 1, thereare shown measured values obtained by measuring the 28 kDa protein,i.e., the marker protein for diagnosing liver disease of the presentinvention, by a conventional method.

Table 2 shows measured values obtained by measuring the 5.9 kDa protein(FDP-E 5,900 Da) and the 7.8 kDa protein (Apo AII 7,800 Da), i.e., themarker proteins for diagnosing liver disease of the present invention,in serum samples from healthy persons.

Table 3 shows average measured values obtained by measuring the 5.9 kDaprotein and the 7.8 kDa protein in the inpatients with alcoholic liverdisease by a protein chip method.

TABLE 1 Changes of clinical examination values in patients withalcoholic liver trouble after abstinence Apo Apo FDP-E Apo AII AST GGTTG AI AII FDP FDP-E 5,900Da 7,800Da U/L U/L mg/dL mg/dL mg/dL μg/mLng/mL AU AU No. 1 On admission 34 542 672 144 37.3 2.99 110.32 11.7 9.9After hospitalized 39 559 246 121 27.8 2.76 135.69 24.2 10.9 for 1 weekAfter hospitalized 25 121 142 79 17.4 1.63 80.20 20.8 6.1 for 3 monthsNo. 2 On admission 13 9 103 88 15.0 3.57 77.28 0 3.1 After hospitalized30 16 98 94 15.1 3.69 136.11 1.0 11.7 for 1 week After hospitalized 15 3173 95 20.8 2.65 126.56 1.8 20.0 for 3 months No. 3 On admission 42 68100 155 38.2 1.37 70.18 0 0 After hospitalized 22 45 91 105 26.0 7.05592.26 0 1.7 for 1 week After hospitalized 18 19 117 104 21.5 15.961257.50 0.7 5.0 for 3 months No. 4 On admission 86 1452 150 234 45.03.39 93.30 0.5 7.9 After hospitalized 21 701 73 112 26.1 5.24 347.94 3.512.6 for 1 week After hospitalized 15 24 68 95 17.7 7.27 516.60 17.721.1 for 3 months No. 5 On admission 24 74 93 95 21.8 0.31 35.11 0.8 2.4After hospitalized 23 64 139 86 21.0 1.36 47.86 4.8 16.1 for 1 weekAfter hospitalized 35 160 154 80 18.5 3.27 71.87 6.2 18.1 for 3 monthsNo. 6 On admission 44 108 141 149 37.7 1.56 42.53 9.2 12.1 Afterhospitalized 25 64 117 98 26.0 3.56 248.95 48.0 21.4 for 1 week Afterhospitalized 17 24 109 83 19.3 8.88 743.54 58.0 20.0 for 3 months No. 7On admission 37 111 119 218 46.5 2.54 90.77 0 0 After hospitalized 27174 77 157 33.7 2.05 91.00 23.0 18.4 for 1 week After hospitalized 19 2681 95 23.3 1.58 50.95 21.7 10.4 for 3 months No. 8 On admission 40 61170 183 33.3 3.87 127.00 1.7 3.3 After hospitalized 24 57 65 113 22.73.17 189.23 0 5.7 for 1 week After hospitalized 22 36 113 101 20.6 2.35105.10 1.8 6.9 for 3 months No. 9 On admission 20 77 244 114 28.4 4.33118.19 0 0.4 After hospitalized 25 65 121 109 24.9 50.13 330.58 0 7.1for 1 week After hospitalized 24 36 260 109 24.0 5.33 270.77 1.7 14.0for 3 months No. 10 On admission 2.0 5.6 After hospitalized 55 232 67 6918.5 13.52 •915.69 8.7 16.1 for 1 week After hospitalized 17 41 54 10720.6 0.87 35.35 1.7 21.4 for 3 months No. 11 On admission 38 81 187 15134.5 2.82 58.02 15.8 19.3 After hospitalized 20 67 98 116 28.1 6.33554.17 56.0 26.4 for 1 week After hospitalized 22 18 74 104 18.1 15.511310.60 52.0 18.6 for 3 months No. 12 On admission 18 37 103 135 24.81.91 43.10 0 11.7 After hospitalized 17 31 110 126 19.6 1.78 68.85 0.86.4 for 1 week After hospitalized 21 32 118 136 22.6 1.03 33.59 1.2 15.0for 3 months No. 13 On admission 18 31 84 198 30.6 1.99 38.14 12.2 9.4After hospitalized 100 23 72 154 22.7 6.74 342.92 32.8 13.9 for 1 weekAfter hospitalized 17 18 80 180 22.6 14.37 1118.50 45.0 15.0 for 3months No. 14 On admission 63 44 333 154 39.5 3.07 96.96 0 3.1 Afterhospitalized 43 39 72 111 27.0 2.50 146.95 0.3 11.0 for 1 week Afterhospitalized 37 26 159 92 23.2 4.73 382.86 0.3 12.1 for 3 months No. 15On admission 49 86 91 146 28.9 1.35 58.93 13.0 7.0 After hospitalized 1979 82 83 23.0 2.29 59.44 28.5 17.7 for 1 week After hospitalized 22 60112 86 22.5 2.99 177.59 51.0 18.0 for 3 months No. 16 On admission 29 8490 162 31.3 3.24 88.54 2.5 1.3 After hospitalized 12 70 118 102 26.61.86 84.94 1.7 10.3 for 1 week After hospitalized 16 15 96 72 17.0 5.84213.87 2.0 13.3 for 3 months

TABLE 2 Levels of 5.9 kDa and 7.8 kDa proteins in healthy persons FDP-EApo A II Number of healthy 5.9 kDa 7.8 kDa persons (n = 12) AU AU No. 159.1 35.1 No. 2 51.7 32.0 No. 3 62.5 42.8 No. 4 63.1 44.0 No. 5 89.295.4 No. 6 58.5 42.2 No. 7 64.6 45.5 No. 8 66.2 44.6 No. 9 23.1 56.9 No.10 55.4 51.7 No. 11 63.7 52.0 No. 12 36.0 24.6

TABLE 3 Average levels of 5.9 kDa and 7.8 kDa proteins FDP-E Apo A II5.9 kDa 7.8 kDa AU AU Healthy persons (n = 12) 57.76 47.23 Immediatelyafter admission 4.34 6.03 After hospitalized for 1 week 14.58 13.00After hospitalized for 3 months 17.73 15.63 Number of patients (n = 16)

As shown in Tables 1 to 3, the decrease of generally used AST and GGTlevels and the recovery of these levels to normal level ranges reflectedthe effect of treatment on liver. However, the blood levels of the 5.9kDa protein and the 7.8 kDa protein increased and clearly indicated theeffect of treatment even when the generally used GGT level was notusable as an indication of the effect of treatment as in the case ofsample No. 5 from a patient who was considered as a nonresponder. TheGGT level does not always correlate with the degree of seriousness of aliver trouble or the cumulative alcohol intake. In addition, the changeof the GGT level after alcohol drinking varies depending on individualsand there are a considerable number of so-called nonresponders who showno increase in the GGT level even after drinking a large volume ofalcohol. Therefore, the novel proteins were considered to be effectivein judging the treatment of these GGT nonresponders.

EXAMPLE 7

Correlation Between Alcohol Intake and the Marker Proteins of thePresent Invention

By the working examples described above, the usefulness of the novelmarkers for diagnosing liver disease found by the present invention wasconfirmed with respect to their specificity. The following experimentwas carried out for further elucidation of the correlation betweenalcohol intake and the markers.

Method

An experiment was carried out by collecting samples only from healthypersons and alcohol-drinking patients whose alcohol intake was certain.In the experiment, these subjects were divided into three experimentalgroups, i.e., a group of healthy persons as nondrinkers, a group ofpatients having an alcohol intake corresponding to 1 go of sake and agroup of patients having an alcohol intake corresponding to 3 go ofsake. For these groups, measurements using protein chip methods,respectively, were carried out and the groups were compared with respectto each of the novel markers. The measuring methods using protein chipswere exactly the same as described in the case of SAXII protein chiparrays in Example 1 and WCXII protein chip arrays in Example 2.

Results

The results are shown in FIG. 7. As a result, it was found that theheights of peaks due to all the novel marker proteins, respectively, fordiagnosing liver disease vary depending on alcohol intake. Here, theresults for, in particular, the 5.9 kDa protein are shown. The 5.9 kDaprotein decreased depending on alcohol intake and it decreased to becomesubstantially undetectable in the experimental group having an alcoholintake of 3 go. That is, it was found that the 5.9 kDa protein varies inamount, depending on alcohol intake and hence is a marker satisfactorilyutilizable for estimation of alcohol intake. It was also found that the5.9 kDa protein is utilizable as a marker for alcohol dependence.

EXAMPLE 8

Preparation of a Monoclonal Antibody

The following experiment was carried out for preparing an anti-5.9 kDaprotein monoclonal antibody by using the completely synthetic 5.9 kDaprotein obtained in Example 5, as an antigen.

(1) Immunization

The completely synthetic 5.9 kDa protein was diluted to a concentrationof 1 mg/ml with phosphate buffer (pH 7.0), and 50 μg (50 μl) of thedilution was thoroughly mixed with 50 μl of Freund's complete adjuvant(WAKO) until emulsification was effected. The suspension thus preparedwas intraperitoneally administered to a Balb/c female mouse aged 6 weeks(Nippon Clear Co., Ltd.) under anesthesia with diethyl ether. After 2weeks, the same amount as above of the completely synthetic 5.9 kDaprotein (50 μg/ml) was mixed with Freund's incomplete adjuvant (WAKO).By exactly the same procedure as in the case of the Freund's completeadjuvant, emulsification was effected to obtain a suspension and themouse was sensitized with the suspension. Two weeks after thesensitization, the same procedure as above was carried out. For thefourth immunization, i.e., final immunization, a dilution of thecompletely synthetic 5.9 kDa protein (50 μg/ml) with phosphate buffer(pH 7.0) was prepared and then administered to the mouse by injectioninto the tail vein.

(2) Establishment of Hybridoma

Three days after the final immunization, the spleen was surgicallyremoved from the mouse sensitized with the synthetic 5.9 kDa protein,under anesthesia with diethyl ether, and was aseptically dispersed toprepare splenocytes. Fusion was carried out according to the method ofKÖhller and Milstein (Nature, 256, 495, 1975). The splenocytes werefused with myeloma cells P3-X63-Ag8-U1 (P3U1) by the use of apoly(ethylene glycol) (PEG4000) (MERK). As to the fusion ratio, thenumber of the splenocytes was 10×10⁷, while the number of the myelomacells P3-X63-Ag8-U1 (P3U1) was 2×10⁷. That is, the fusion ratio of thesplenocytes to the myeloma cells was 5:1. The fused cells were dispersedin 10% FCS (INVITROGEN) α-MEM (IRVINE) HAT (Cosmo Bio Co., Ltd.), seededinto a 96-wells microtiter culture plate (Sumitomo Bakelite Co., Ltd.)and then cultured under conditions of 37° C. and 5% CO₂.

(3) Screening

After about 2 weeks, the growth of colonies was confirmed and screeningwas conducted. A method for conducting the screening is described below.For producing a plate for the screening, the synthetic 5.9 kDa proteinpurified in the above working example was dissolved in phosphate bufferand fed into a 96-well plate (Nunc) in an amount of 1 μg/100 μl/well.The plate was allowed to stand at 4° C. for two nights and then washedthree times with phosphate buffer containing 0.05% Tween 20. Each wellwas fed with 200 μl of 1.5% BSA solution in order to inhibit anonspecific reaction, and the plate was allowed to stand overnight at 4°C. After the thus completed plate was washed three times with phosphatebuffer containing 0.05% Tween 20, 100 μl of the culture supernatant wasreacted in each well and the plate was further washed. Then, HRP-labeledanti-mouse immunoglobulin antibody (Zymed), a secondary antibody wasadded to carry out the reaction. After washing, 100 μl of a 3 mg/mlcolor-producing solution of o-phenylenediamine (OPD) (Nacalai tesque), acolor-producing substrate for HRP, in citric acid was added to each wellto cause coloration for a definite period. Then, 100 μl of 1N sulfuricacid was added to each well as a terminating solution and absorbance wasmeasured at a measuring wavelength of 492 nm. Clones found to bepositive by the above procedure were subjected to recloning by alimiting dilution method, and the resulting supernatants were checkedagain.

(4) Confirmation of Antibodies

Two clones, i.e., clones CN-1 and CN-2 were selected as clones which hadrecognized the synthetic 5.9 kDa protein, by confirming their reactivitywith the synthetic 5.9 kDa protein by ELISA. Table 4 shows the resultsof assaying antibodies produced by these clones, by the use of amonoclonal antibody typing kit (Amersham Pharmacia Biotech).

TABLE 4 Characteristics of monoclonal antibodies Hybridoma Class Lightchain Clone CN-1 IgM κ Clone CN-2 IgM κ(5) Preparation and Purification of the Monoclonal Antibodies

To a Balb/c female mouse aged 10 weeks (Nippon Clear Co., Ltd.) twoweeks after administration of 0.5 ml of pristane (Aldrich) to the mousewere intraperitoneally administered 1×10⁷ cells of each of thehybridomas CN-1 and CN-2 obtained. After about 2 weeks, ascitesaccumulated in the abdominal cavity of the mouse was surgicallycollected under anesthesia with diethyl ether. As a result ofconfirmation by the ELISA method adopted in the screening, by the use ofthe ascites stepwise diluted as a sample, it was found that the ascitescontained a high concentration of the monoclonal antibody. The asciteswas treated with 40% ammonium sulfate and dialyzed against PBS, and thenthe monoclonal antibodies CN-1 and CN-2 were purified by the use ofS-300. As a result, a single band was confirmed at a molecular weight ofabout 900,000 when each of the monoclonal antibodies CN-1 and CN-2 hadnot been reduced, and two bands were confirmed at molecular weights ofabout 70,000 and 25,000 when each of them had been reduced withmercaptoethanol. The amount of each of the purified antibodies CN-1 andCN-2 was about 10 mg or more per mouse, namely, it was sufficient forindustrial utilization.

EXAMPLE 9

Measurement of the 5.9 kDa Protein by EIA Method

There was investigated the possibility of measurement of the 5.9 kDaprotein in a sample by ELISA method (EIA method) by the use of the twomonoclonal antibodies CN-2 and CN-1 against the 5.9 kDa protein as aprimary antibody and a secondary antibody, respectively.

(1) Method

For preparing a plate for ELISA, the primary antibody CN-2 was dissolvedin phosphate buffer (pH 6.7) and fed into a 96-well plate (Nunc) in anamount of 1 μg/100 μl/well. The plate was allowed to stand at 4° C. fortwo nights and then washed three times with phosphate buffer containing0.05% Tween 20. Each well was fed with 200 μl of 1.5% BSA solution inorder to inhibit a nonspecific reaction, and the plate was allowed tostand overnight at 4° C. After the thus completed plate was washed threetimes with phosphate buffer containing 0.05% Tween 20, 100 μl of variousconcentrations of the synthetic 5.9 kDa protein was added to each wellas a reference standard and the reaction was carried out at roomtemperature for 1 hour. After completion of the reaction, the plate waswashed three times with phosphate buffer containing 0.05% Tween 20, andHRP-labeled CN-1 antibody as secondary antibody was added to carry outthe reaction. After washing, 100 μl of a 3 mg/ml color-producingsolution of o-phenylenediamine (OPD) (Nacalai tesque), a color-producingsubstrate for HRP, in citric acid was added to each well to causecoloration for a definite period. Then, 100 μl of 1N sulfuric acid wasadded to each well as a terminating solution and absorbance was measuredat a measuring wavelength of 492 nm.

(2) Results

On the basis of coloration values measured for the variousconcentrations of the synthetic 5.9 kDa protein as described above, astandard curve for measuring the synthetic 5.9 kDa protein was preparedto obtain the result shown in FIG. 8. As can be seen from FIG. 8,absorbance increased depending on the concentration of the synthetic 5.9kDa protein. That is, it was found that since the monoclonal antibodiesCN-1 and CN-2 capable of recognizing the 5.9 kDa protein were differentin epitope, the 5.9 kDa protein could be measured by using theseantibodies in sandwich ELISA method.

INDUSTRIAL APPLICABILITY

As concretely described above, the marker proteins for diagnosing liverdisease of the present invention may be used as an indication foraccurately investigating the effect of judgment for the treatment of apatient with liver disease due to drinking to grasp the condition of thepatient, even when the GGT level of the patient is high owing to a causeother than drinking as in the case of, for example, a nonresponderhaving a low reactivity with conventional markers for hepatitis (e.g.GGT), fatty liver accompanying corpulence, or a person who commonly usesa certain medicine. That is, GGT has a low specificity because it isincreased by a cause other than drinking, such as a viral chronic livertrouble, corpulence, or continuous use of a certain medicine. On theother hand, the marker proteins for diagnosing liver disease of thepresent invention are advantageous in that their specificity is hopefulbecause their levels do not vary even in the case of viral livercirrhosis. It is also found that these marker proteins are characterizedby permitting easy handling of samples and giving a high reproducibilityof measurement because substances to be measured in this case arepeptides which are not dependent on biochemical enzymatic function. Thediagnosis method of the present invention also permits diagnosis of theprobability of the onset of a liver disease in a habitual drinker or aproblem drinker and diagnosis of a liver disease caused by drinking,such as hepatitis, liver cirrhosis or the like. Moreover, it alsopermits diagnosis of, for example, the progress of treatment of such aliver disease. The diagnosis method of the present invention is suitablefor diagnosis of, in particular, an alcoholic liver trouble, alcoholdependence or the like. In the diagnosis method of the presentinvention, diagnosis may be carried out by measuring the marker proteinsby a generally used EIA method, immuno-chromatography, test paper or thelike. The fact that the diagnosis can easily be carried out by such agenerally used method is considered to be of great significance from theviewpoint of future preventive medicine in consideration of the presentpopulation of liver disease sufferers.

1. An isolated protein consisting of the amino acid sequence shown asSEQ ID NO: 1 in the sequence listing.